New advances in genetic diagnoses and Diagnoses in Rare Diseases
Carlos Bacino, M.D Dept. of Molecular and Human Genetics Baylor College of Medicine and Texas Children’s Hospital Houston, Texas, USA
Disclosures
• Employer: Baylor College of Medicine and Medical Director for Baylor Genetics Cytogenetics Laboratory • Consultant: Best Doctors Inc.
• Research funding: • Pfizer, Ascendis and BioMarin for clinical research studies in
achondroplasia • Hoffman-La Roche for clinical research studies in Angelman syndrome
Learning Objectives
• Review the current tools used in genetic/genomic testing • Discuss the advantages and
limitations of whole exome sequencing • Describe the use of RNA
sequencing in clinical research and potential clinical applications
20,000
History of Genetic Testing
• 1956 - Humans have 46 chromosomes - Tjio • 1959 - Trisomy 21 is the cause of Down
syndrome - Lejeune • Trisomy D (13), trisomy E (18), cri-du-chat
deletion first identified • 1968 - First chromosome banding - Cassperson • 1981 - Attainment of 2000 band karyotype –
Yunis • 1990’s - FISH • 2005 - CGH/Chromosome Microarray 2005 • 2015 - Exome and Genome sequencing • 2020 – RNA sequencing
G-banding: chromosomes subjected to trypsin digestion
46,XX
Chromosomal Microarray Analysis (CMA)
• CMA is an array-based genomic hybridization methodology that allows for analysis of the Copy Number Variations (CNV) within chromosomes for a large number of genetic disorders • With a single test, CMA can identify the
abnormalities that are detectable by routine chromosome analysis, FISH and MLPA analyses • Different microarrays: whole genome arrays
and SNP arrays
CMA [average resolution ~30kb, whole genome]
FISH - Fluorescent in situ hybridization [40 to 250 kb per probe, single site]
Genomic Resolution Conventional karyotype [4-5 Mb, whole genome]
Chromosome Microarray 400,000 oligonucleotides
Genomic Disorders • Genomic disorders result from chromosomal and/or genomic imbalance • Large number of well delineated genomic disorders • Over 40 well recognized syndromes • Altered gene dosage for any extensive chromosomal or genomic region is likely to
result in a clinical abnormality, the phenotype will reflect: • Haploinsufficiency of gene/s • Copy number gains: duplication/triplication: gene overexpression • Contiguous gene syndromes caused by the cumulative effect of multiple genes
Chromosomal Microarray Analysis (CMA)
Diagnosis of Microdeletions
• Routine chromosome studies achieve 400-500 bands. • Microdeletions are losses of DNA below the level of • resolution by G-banding
A 3 Mb deletion contains 10-100 genes Only a few genes are likely to be dosage sensitive Sometimes a single gene may account for most features Multiple genes deleted = contiguous gene deletion syndromes
What do Arrays Detect? Microdeletions/Microduplications
Routine cytogenetic studies achieve 450-500 bands • Cannot detect deletions or duplications 5-10 Mb (1 MB = 1 million base pairs) • Deletion & Duplications < 5 Mb requires molecular techniques • Occur via recombination mechanisms • Often resulting from recombination between non-allelic low copy repeat
(LCR) sequences • Reciprocal deletions and duplications expected to occur with equal frequency
Deletion Reciprocal duplication
Scheme for the Non-allelic homologous recombination (NAHR) between low copy repeats (LCRs)
Limitations of Microarray Methodology
• Cannot detect balanced rearrangements • Reciprocal translocations • Inversions • Insertions
• Does not provide information regarding the location of additional copy • Translocation • Marker • Duplication in same location or inserted somewhere else in the genome
Novel applications of array comparative genomic hybridization in molecular diagnostics. Sau W Cheung, Weimin Bi. PMID: 29848116
Microdeletion & Reciprocal Microduplication Chromosome
15q11-13 PWS/ Angelman
stenosis (elastin deletion) • Mild intellectual disability
with IQ’s up to 80 • Typical behaviors:
overfriendliness, attention deficit disorder
• Variable degree of language delay and developmental delay
• Autism-spectrum behavioral abnormalities
Speech delay and autism spectrum behaviors are frequently associated with duplication of the 7q11.23 Williams-Beuren syndrome region. Berg JS et al. Genet Med. 2007 Jul;9(7):427-41. PMID: 17666889
7q11.23 Duplications
• Developmental Delay (mild to moderate) • Speech delay • Dysmorphic Features (mild and variable) • Behavioral abnormalities:
• Autistic spectrum behaviors: social impairment, stereotypical or repetitive behaviors
Nextgen sequencing
The bases of a small fragment of DNA are sequentially identified from signals emitted as each fragment is re- synthesized from a DNA template strand
NGS extends this process across millions of reactions in a massively parallel fashion
This technique enables rapid sequencing of large stretches of DNA base pairs spanning entire genomes, producing hundreds of gigabases of data in a single sequencing run
How do we study rare disease patients? What is exome and a genome?
• Exome is the sum of exons: short, functionally important sequences of DNA which represent the regions in genes that are translated into protein and make up 1.5 - 2 % of the whole genome
• Genome is the complete set of genes and genetic material present in a cell or organism
Exome corresponds to 1.5 – 2% of the genome
WHAT IS AN EXOME AND A GENOME
Use of next-generation sequencing and other whole genome strategies to dissect neurological disease. Bras J, Guerreiro R, Hardy J. Nat Rev Neurosci. 2012 Jun 20;13(7):453-64. Review. PMID: 22714018
Use of next-generation sequencing and other whole genome strategies to dissect neurological disease. Bras J, Guerreiro R, Hardy J. Nat Rev Neurosci. 2012 Jun 20;13(7):453-64. Review. PMID: 22714018
ES Strengths and advantages
• WES is an efficient method to discover the genetic cause of diseases since it analyzes exons of tens of thousands of genes at the same time • Cost-effective and less data generated
as compared to WGS • Improve preventive care through
identification of medically actionable result • The exome is the well-known part of
the genome
ES Limitations
Due to technology limitations does not cover 100% of the exome: ~90%-95% depending on methodology.
Depth of coverage varies across exome platforms
Not detected:
• Triplet repeat expansions • Large deletions & duplications • Deep intronic mutations • Regulatory mutations • Genes that have closely related pseudogenes (processed and
unprocessed) are not uniquely captured by this method • Mutations in UTR regions
Exomes vs. Genomes
ES and GS allow diagnosis in 30% and can go up to 40-50% of patients depending how performed and analyzed (single versus trio)
Genome Sequencing is still more widely used in research. Reporting mostly limited to exome contents but may detect over 50%
Rare Diseases
• What is a rare disease? • A disease with prevalence of <200,000 people in
the US affecting 1:1,500
• Many are genetic diseases • How many rare diseases are there? • Around 7,000
• How many individuals have a rare disease? • ~30 million Americans (~8-10% of the
population) • Only a few hundred rare diseases have FDA-
approved therapies
NIH/ORDR – Office of Rare Diseases Research. NCATS – National Center for Advancing Translational Sciences
RNA Sequencing
• A recent study at BCM showed increased diagnosis detection through the UDN • 12% of cases solved with transcriptome
analysis (14 out of 114) • All had exome and/or genome sequencing • The approach to RNA-seq analysis focuses
on splicing and the expression levels of genes • Tissues vary and have different expression
Transcriptome-directed analysis for Mendelian disease diagnosis overcomes limitations of conventional genomic testing. Murdock DR et al. Clin Invest. 2021 Jan 4;131(1):e141500. doi: 10.1172/JCI141500. PMID: 33001864
Case 1 - Clinical Presentation • 3-year-old male with multiple congenital anomalies. • Hypospadias, SNHL with cochlear implants, failure to thrive, congenital heart defects
(VSD/PDA), and a single butterfly vertebra. • The pregnancy complicated by IUGR. • Prenatal exposures include VAPE e-cigs, Paxil for anxiety at around 30 weeks and
gabapentin 100 mg. • Born NSVD. Apgar scores: 1, 4 and 6 at 1, 5 and 10 minutes. • Birth Weight 1.721 kg, length 42 cm, and FOC 30 cm 3rd, all below the percentile and
noted to have dysmorphic features • Feeds via G-tube. Constipation. • Rolls over at 12 months, not sitting at 3 years
Family history
• Half brother through the mother has VACTERL. • Father's sister with cleft lip. • Half sibling died in utero at 36 weeks with fetal anomalies (was 2 lbs
at the 36 weeks, autopsy showed that he had a cardiac defect, a lung defect and gastroschisis) • Mother's aunt with Down Syndrome. • Bipolar and depression in through family.
• Small and dysmorphic. His weight %ile: <1 %ile (Z= -3.84), FOC: <1 %ile (Z= -4.59).
• Microbrachycephaly, deep set eyes, sparse eyebrows. Midface hypoplasia. Broad nose. Ears are borderline low set, small and with thick helices. Mouth small, thin lips. Palate is high. Long philtrum. Broad neck. Nipples are low set.
• Slender and built appearance • Small phallus, hypospadias.
Physical Exam
• Hands show fifth finger clinodactyly, small nails, thumbs are proximally implanted.
• Tips of 3,4,5 digits are broad.
• Feet show short toes 2nd to 5th. Syndactyly between 3-4th toes.
• Limbs and body with reticular vascular patchy appearance
Physical Exam
Ancillary Studies
• Renal U/S: Ectopic pelvic left kidney. • ECHO: VSD, PDA • CXR: T10 Vertebral butterfly vertebrae • Head U/S: Structurally normal. • CMA-HR+SNP(V10.2) Normal • Sterol panel normal • Two exome studies and genome sequencing showed two changes
associated with carrier status for two autosomal recessive disorders
RNA-seq analysis detected a 50% reduction in the patient expression of PQBP1 compared with controls in whole blood.
Reanalysis of GS data revealed a hemizygous deep intronic variant in PQBP1 (c.180-306G>A) inherited from the mother that activated a cryptic splice donor near the variant site
The RNA-seq pipeline also detected an abnormal splicing pattern that resulted in an out-of-frame pseudoexon between exons 3 and 4, as well as more distal intron retention (Figure 4C).
Defects in PQBP1 cause Renpenning syndrome an X-linked ID syndrome characterized by males with microcephaly, short stature, cardiac and renal anomalies, small testes, and dysmorphic features.
Transcriptome-directed analysis for Mendelian disease diagnosis overcomes limitations of conventional genomic testing. Murdock DR et al. Clin Invest. 2021 Jan 4;131(1):e141500. doi: 10.1172/JCI141500. PMID: 33001864
Renpenning Syndrome - Summary
The Renpenning syndrome spectrum: new clinical insights supported by 13 new PQBP1-mutated males. Germanaud D, Rossi M, Bussy G, Gérard D, Hertz-Pannier L, Blanchet P, Dollfus H, Giuliano F, Bennouna-Greene V, Sarda P, Sigaudy S, Curie A, Vincent MC, Touraine R, des Portes V.Clin Genet. 2011 Mar;79(3):225-35. PMID: 20950397
Renpenning Syndrome - Summary
dysmorphic features. • Early mild height growth impairment with
significant lean body build and scanty subcutaneous fat and rather thin habitus, often attributed to feeding difficulties. • Upper back progressive muscular atrophy. • MCP ankylosis of the thumb • Velar dysfunction
7-year-old female with intellectual disability, dysmorphic features, and seizures.
History of hypotonia evident at 2 months.
Medical history is also significant for asthma, laryngotracheomalacia, recurrent OM and upper airway infections, had PE tubes and T&A
High hyperopia (+5.50 OU) and accommodative esotropia.
She started with seizures with focal motor onset at approximately 5 years of age. Seizures are well controlled with Keppra.
Picture at 9 years
Case 2 - Clinical Presentation
• Normocephalic, normal growth (anthropometric measurements between 75-90 % ile)
• Hypertelorism with slightly upslanting palpebral fissures, telecanthus, mild epicanthal folds,
• Thin vermillion and fleshy lower lip, long philtrum, broad nose with tubular shape, possible macrodontia (wide incisors)
• Broad halluces and soft palms • Scoliosis
Clinical Course and Testing
• Developmentally she showed milestone delays. Sits up at 8 months, crawls at 19 months, pulls to stand at 18 months, walks at 21 months. • Her IQ is in the 50s • Vocabulary includes 20-30 words and she currently
only combines 2-3 words with indistinct speech. • Negative trio GeneDx WES 2015, reanalyzed 2017 • Negative CMA 2012
Tissue Gene OMIM Mode pLI Haplo Triplo FB TPM
WB TPM pValue zScore foldChange
FB KANSL1 yes AD 1 3 0 11.1 8.6 1.8713E-08 -6.06 0.52
RNA Pathway Analysis De Novo RNAseq Expression
RNAseq + WES
Hypothesis
De novo small KANSL1 deletion (5UTR-exon 2) missed on CMA
NMD occurring à KANSL1 expression reduced by half à KdVS
Koolen-de Vries syndrome (KdVS)
• Heterozygous 500-650 kb deletion at chromosome 17q21.31 that includes KANSL1 or a heterozygous intragenic pathogenic variant in KANSL1 • Developmental delay, learning disability,
hypotonia, epilepsy, heart defects, kidney/urological anomalies, dysmorphic features • Behavior in most is described as friendly,
amiable, and cooperative
Growth 75-95th %ile for height, weight, FOC
Koolen-de Vries syndrome (KdVS) • Developmental delay/intellectual disability (mild-to-moderate) • Neonatal/childhood hypotonia • Dysmorphic features, • Behavioral features (described as friendly, amiable, and cooperative). • Speech and language delay (100%) • Epilepsy (~33%) • Congenital heart defects (25%-50%) • Renal and urologic anomalies (25%-50%) • Cryptorchidism (71% of males) • Hypermobility of the joints and/or joint dislocation/dysplasia • Deformities of the spine and/or feet • Hypermetropia
Koolen-de Vries syndrome (KdVS) – Gene Reviews
• Upslanted palpebral fissures • Blepharophimosis • Epicanthus • Ptosis • Pear-shaped nose • Bulbous nose • Large/protruding ears
Summary
• Genetic diagnoses went from chromosome to genomic and then single gene disorders • Array technology aid with the discovery of many
new genomic disorders • The adding of arrays and next generation
sequencing has changed the landscape of diagnoses in rare diseases • NGS allowed exome and genome sequencing to
increase detection in patients with intellectual disabilities and multiple congenital anomalies • RNA sequencing is a promising tool that will be
soon added to the toolbox of clinical diagnoses.